The arterial wall is a complex macromolecular structure. One of the major elements of these structures is the scaffold that provides the strength and flexibility to perform the task in hand either retaining the blood in vessels against the arterial pressure or maintaining pressure via the function of coronary valves. In the last several years it has become apparent that the actual microstructure and composition of these macromolecules could influence the progress of different disease states most notably atherosclerosis and valve calcification. To gain a better understanding of this process, we have embarked on studies to understand the fine structure of the macromolecules in arterial vascular bed using a novel optical imaging technique that relies on the non-linear excitation (NLE) of collagen and elastin to provide sub-micron images of their structure in unfixed fresh samples together with direct monitoring of water permeation through the wall using Coherent anti-Stokes Raman Scattering(CARS). These results were published demonstrating that the major barrier to water permeation was the basolateral membrane of the endothelial cells. While the apical membrane (facing the blood) was highly permeable to water. We hypothesized that apical membrane AQP-1 was the major source of high water permeability in the apical membrane. New studies included: 1) One question raised in the review of our earlier work was whether this could be studied on vascular endothelial cells in culture where more control and better optics could be realized. We explored this possibility with Dr. Huang at Cambridge University to attempt to develop an in vitro cultured vascular endothelial cell preparation with appropriate distribution of proteins in the apical and basolateral membranes. These studies revealed that the culture systems do not generate adequate water impermeability to conduct these studies. 2) Together with Dr. Dora at the University of Oxford we are expanding our studies on perfused vessels to include the mouse internal cerebral arteries. The water handling in these vessels in the volume restricted structure of the skull was particularly interesting from both a clinical and basic sciences perspective. We have found that these smaller vessels generate far superior images of the artery wall and confirmed our findings in the rate mesenteric arteries. We re-established a AQP-1 knockout mouse to test our hypothesis that the apical membrane is highly permeable to water due the presence of AQP-1. Using the newly developed superior cerebral artery model we found no difference in the control and knockout mouse suggesting that AQP-1 is not the major element in the high water permeability of the apical membrane. We are now exploring other possibilities.